Eukaryotic cells are faced with a dual challenge of packaging all of their genetic information into chromatin (made up of histone/DNA complexes called nucleosomes) while at the same time making this information selectively accessible to accommodate key genomic processes. Cells deal with this problem by establishing a set of histone post translational modifications (PTMs) that regulate the dynamic transition between open and closed chromatin states. These modifications may promote chromatin state transitions through alterations of biophysical properties of the chromatin fiber or recruitment of effector molecules which in turn interpret (read) or change (write or erase) the modifications thus altering the chromatin state. Histone phosphorylation is one specific example of a critically important type of PTM. Most of the well-studied examples of histone phosphorylation occur on serine and threonine residues. One of the best representative examples is g-H2A.X (Ser139phos) that marks chromatin for double strand breaks (DSBs) and is vital for proper repair of DSBs as well as maintenance of genomic stability and therefore is a critical component in cancer development. The Allis lab has been instrumental in defining several phosphorylations on all of the core histones which may act independently or as part of PTM motifs containing multiple modifications (i.e. acetyl/phos or methyl/phos). These discoveries have been instrumental for elucidating such key cellular mechanisms as DNA repair, mitosis and transcription. There is, however, another class of histone phosphorylation that has eluded characterization because of its labile nature. Development of novel analysis tools allowed us to overcome previous challenges and make important headway in studying histidine phosphorylation in the chromatin context. Histidine on histone H4, one of the four histones making up the core histone octamer, has been shown to be phosphorylated and associated with active transcription. One of the main goals of the work outlined in this proposal is to gain mechanistic understanding of the regulatory machinery involved in depositing this modification, its effect on important biological processes such as transcription and replication, or disease progression. A complimentary goal is to determine the effect of this modification on the structural properties of the chromatin fiber. Designer chromatin, an invaluable tool for studying chromatin modifications and properties will be used alone and in conjunction with the cell-free transcription assay system to test the functional outputs of various chromatin states. Overall the work proposed here has exciting potential to elucidate a major regulatory mechanism that controls the transition between chromatin states as well as various biological processes.